RESUMO
The mammalian sterile 20-like (MST) kinases are central constituents of the evolutionary ancient canonical Hippo pathway regulating cell proliferation and survival. However, perhaps surprisingly, MST1 deficiency in human patients leads to a severe combined immunodeficiency syndrome with features of autoimmune disease. In line with this, Mst1-deficient mice exhibit severe defects in lymphocyte and neutrophil functions as well as disturbed intracellular vesicle transport. These findings spurred research on the noncanonical functions of MST1 in leukocytes. Here, we summarise the latest findings on this topic and discuss MST1 as a critical regulator of various leukocyte functions.
Assuntos
Doenças Autoimunes/genética , Leucócitos/metabolismo , MAP Quinase Quinase Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Imunodeficiência Combinada Severa/genética , Animais , Evolução Biológica , Via de Sinalização Hippo , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Transdução de SinaisRESUMO
Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.
Assuntos
Neutrófilos , Quinases da Família src , Animais , Membrana Basal , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas , Quinases da Família src/genéticaRESUMO
A change regarding the extent of adhesion - hereafter referred to as adhesion plasticity - between adhesive and less-adhesive states of mammalian cells is important for their behavior. To investigate adhesion plasticity, we have selected a stable isogenic subpopulation of human MDA-MB-468 breast carcinoma cells growing in suspension. These suspension cells are unable to re-adhere to various matrices or to contract three-dimensional collagen lattices. By using transcriptome analysis, we identified the focal adhesion protein tensin3 (Tns3) as a determinant of adhesion plasticity. Tns3 is strongly reduced at mRNA and protein levels in suspension cells. Furthermore, by transiently challenging breast cancer cells to grow under non-adherent conditions markedly reduces Tns3 protein expression, which is regained upon re-adhesion. Stable knockdown of Tns3 in parental MDA-MB-468 cells results in defective adhesion, spreading and migration. Tns3-knockdown cells display impaired structure and dynamics of focal adhesion complexes as determined by immunostaining. Restoration of Tns3 protein expression in suspension cells partially rescues adhesion and focal contact composition. Our work identifies Tns3 as a crucial focal adhesion component regulated by, and functionally contributing to, the switch between adhesive and non-adhesive states in MDA-MB-468 cancer cells.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Adesão Celular/genética , Tensinas/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Adesões Focais/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , HumanosRESUMO
Cutaneous immunity represents a crucial component of the mammalian immune response. The presence of a large array of commensal microorganisms along with a myriad of environmental stresses necessitates constant immuno-surveillance of the tissue. To achieve a perfect balance between immune-tolerance and immune-activation, the skin harbors strategically localized immune cell populations that modulate these responses. To maintain homeostasis, innate and adaptive immune cells assimilate microenvironmental cues and coordinate cellular and molecular functions in a spatiotemporal manner. The role of lymphoid cells in cutaneous immunity is gaining much appreciation due to their important roles in regulating skin health and pathology. In this review, we aim to highlight the recent advances in the field of cutaneous lymphoid biology.
Assuntos
Linfócitos/imunologia , Pele/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa , Animais , Microambiente Celular , Homeostase , Humanos , Tolerância Imunológica , Imunidade Inata , Memória Imunológica , Receptores de Antígenos de Linfócitos T gama-delta/metabolismoRESUMO
OBJECTIVE: Platelet function has been intensively studied in the adult organism. However, little is known about the function and hemostatic capacity of platelets in the developing fetus as suitable in vivo models are lacking. APPROACH AND RESULTS: To examine fetal platelet function in vivo, we generated a fetal thrombosis model and investigated light/dye-induced thrombus formation by intravital microscopy throughout gestation. We observed that significantly less and unstable thrombi were formed at embryonic day (E) 13.5 compared with E17.5. Flow cytometry revealed significantly lower platelet counts in E13.5 versus E17.5 fetuses versus adult controls. In addition, fetal platelets demonstrated changed activation responses of surface adhesion molecules and reduced P-selectin content and mobilization. Interestingly, we also measured reduced levels of the integrin-activating proteins Kindlin-3, Talin-1, and Rap1 during fetal development. Consistently, fetal platelets demonstrated diminished spreading capacity compared with adults. Transfusion of adult platelets into the fetal circulation led to rapid platelet aggregate formation even in young fetuses. Yet, retrospective data analysis of a neonatal cohort demonstrated no correlation of platelet transfusion with closure of a persistent ductus arteriosus, a process reported to be platelet dependent. CONCLUSIONS: Taken together, we demonstrate an ontogenetic regulation of platelet function in vivo with physiologically low platelet numbers and hyporeactivity early during fetal development shedding new light on hemostatic function during fetal life.
Assuntos
Plaquetas/metabolismo , Hemostasia , Ativação Plaquetária , Trombose/sangue , Animais , Moléculas de Adesão Celular/sangue , Bases de Dados Factuais , Modelos Animais de Doenças , Permeabilidade do Canal Arterial/sangue , Feminino , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Recém-Nascido de muito Baixo Peso , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Adesividade Plaquetária , Transfusão de Plaquetas , Nascimento Prematuro/sangue , Estudos Retrospectivos , Transdução de Sinais , Trombocitopenia/sangueRESUMO
ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Migração Transendotelial e Transepitelial , Animais , Proteínas Ativadoras de GTPase/deficiência , Camundongos , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/metabolismoRESUMO
OBJECTIVE: Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure. APPROACH AND RESULTS: Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue. CONCLUSIONS: Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases.
Assuntos
Células Endoteliais/metabolismo , Selectina L/metabolismo , Migração e Rolagem de Leucócitos , Monócitos/metabolismo , Neutrófilos/metabolismo , Selectina-P/metabolismo , Peritonite/metabolismo , Migração Transendotelial e Transepitelial , Animais , Receptor 1 de Quimiocina CX3C , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/imunologia , Hemodinâmica , Receptores de Hialuronatos/metabolismo , Mediadores da Inflamação/metabolismo , Ligantes , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcirculação , Microvasos/imunologia , Microvasos/metabolismo , Microvasos/fisiopatologia , Monócitos/imunologia , Neutrófilos/imunologia , Peritonite/genética , Peritonite/imunologia , Peritonite/fisiopatologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
OBJECTIVE: The polarity protein Scrib is highly expressed in endothelial cells and is required for planar cell polarity. Scrib also facilitates recycling of integrin α5 to the plasma membrane. Because integrin α5 signals the presence of the inflammatory matrix protein fibronectin, we hypothesized that Scrib contributes to endothelial inflammatory signaling. APPROACH AND RESULTS: Cytokine treatment of human umbilical vein endothelial cells induced an inflammatory response as evident by the induction of vascular cell adhesion molecule-1 (VCAM-1). Downregulation of Scrib greatly attenuated this effect. In endothelial-specific conditional Scrib knockout mice, in vivo lipopolysaccharide treatment resulted in an impaired VCAM-1 induction. These effects were functionally relevant because Scrib small interfering RNAs in human umbilical vein endothelial cells attenuated the VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α. In vivo, tamoxifen-induced endothelial-specific deletion of Scrib resulted in a reduced VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α in the mouse cremaster model. This effect was specific for Scrib and not mediated by other polarity proteins. Moreover, it did not involve integrin α5 or classic pathways supporting inflammatory signaling, such as nuclear factor κ light chain enhancer of activated B-cells or MAP kinases. Co-immunoprecipitation/mass spectrometry identified the zinc finger transcription factor GATA-like protein-1 as a novel Scrib interacting protein. Small interfering RNA depletion of GATA-like protein-1 decreased the tumor necrosis factor-α-stimulated VCAM-1 induction to a similar extent as loss of Scrib did. Silencing of Scrib reduced GATA-like protein-1 protein, but not mRNA abundance. CONCLUSIONS: Scrib is a novel proinflammatory regulator in endothelial cells, which maintains the protein expression of GATA-like protein-1.
Assuntos
Artérias Carótidas/metabolismo , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA/genética , Animais , Western Blotting , Artérias Carótidas/patologia , Células Cultivadas , Modelos Animais de Doenças , Fator de Transcrição GATA1/biossíntese , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
In adult mammals, leukocyte recruitment follows a well-defined cascade of adhesion events enabling leukocytes to leave the circulatory system and transmigrate into tissue. Currently, it is unclear whether leukocyte recruitment proceeds in a similar fashion during fetal development. Considering the fact that the incidence of neonatal sepsis increases dramatically with decreasing gestational age in humans, we hypothesized that leukocyte recruitment may be acquired only late during fetal ontogeny. To test this, we developed a fetal intravital microscopy model in pregnant mice and, using LysEGFP (neutrophil reporter) mice, investigated leukocyte recruitment during fetal development. We show that fetal blood neutrophils acquire the ability to roll and adhere on inflamed yolk sac vessels during late fetal development, whereas at earlier embryonic stages (before day E15), rolling and adhesion were essentially absent. Accordingly, flow chamber experiments showed that fetal EGFP(+) blood cells underwent efficient adhesion only when they were harvested on or after E15. Fluorescence-activated cell sorter analysis on EGFP(+) fetal blood cells revealed that surface expression of CXCR2 and less pronounced P-selectin glycoprotein ligand-1 (PSGL-1) begin to increase only late in fetal life. Taken together, our findings demonstrate that inflammation-induced leukocyte recruitment is ontogenetically regulated and enables efficient neutrophil trafficking only during late fetal life.
Assuntos
Movimento Celular/imunologia , Sistema Imunitário/embriologia , Leucócitos/citologia , Microvasos/embriologia , Saco Vitelino/embriologia , Animais , Adesão Celular/imunologia , Eritroblastos/citologia , Feminino , Sangue Fetal/citologia , Proteínas de Fluorescência Verde/metabolismo , Sistema Imunitário/citologia , Migração e Rolagem de Leucócitos/imunologia , Leucócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Microvasos/citologia , Microvasos/imunologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Selectina-P/metabolismo , Gravidez , Receptores de Interleucina-8B/metabolismo , Saco Vitelino/irrigação sanguínea , Saco Vitelino/citologiaRESUMO
Short-chain fatty acids (SCFAs) are produced by microbial fermentation of dietary fiber in the gut. Butyrate is a particularly important SCFA with anti-inflammatory properties and is generally present at lower levels in inflammatory diseases associated with gut microbiota dysbiosis in mammals. We aimed to determine if SCFAs are produced by the zebrafish microbiome and if SCFAs exert conserved effects on zebrafish immunity as an example of the non-mammalian vertebrate immune system. We demonstrate that bacterial communities from adult zebrafish intestines synthesize all three main SCFA in vitro, although SCFA were below our detectable limits in zebrafish intestines in vivo. Immersion in butyrate, but not acetate or propionate, reduced the recruitment of neutrophils and M1-type pro-inflammatory macrophages to wounds. We found conservation of butyrate sensing by neutrophils via orthologs of the hydroxycarboxylic acid receptor 1 (hcar1) gene. Neutrophils from Hcar1-depleted embryos were no longer responsive to the anti-inflammatory effects of butyrate, while macrophage sensitivity to butyrate was independent of Hcar1. Our data demonstrate conservation of anti-inflammatory butyrate effects and identify the presence of a conserved molecular receptor in fish.
Assuntos
Anti-Inflamatórios/farmacologia , Butiratos/metabolismo , Butiratos/farmacologia , Microbioma Gastrointestinal/fisiologia , Macrófagos/imunologia , Neutrófilos/imunologia , Acetatos/farmacologia , Animais , Fibras na Dieta/metabolismo , Disbiose/microbiologia , Macrófagos/efeitos dos fármacos , Masculino , Neutrófilos/efeitos dos fármacos , Propionatos/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ferimentos e Lesões/imunologia , Peixe-Zebra/embriologia , Peixe-Zebra/imunologiaRESUMO
The Mediterranean diet, rich in olive oil, is beneficial, reducing the risk of cardiovascular diseases and cancer. Olive oil is mostly composed of the monounsaturated fatty acid omega-9. We showed omega-9 protects septic mice modulating lipid metabolism. Sepsis is initiated by the host response to infection with organ damage, increased plasma free fatty acids, high levels of cortisol, massive cytokine production, leukocyte activation, and endothelial dysfunction. We aimed to analyze the effect of omega-9 supplementation on corticosteroid unbalance, inflammation, bacterial elimination, and peroxisome proliferator-activated receptor (PPAR) gamma expression, an omega-9 receptor and inflammatory modulator. We treated mice for 14 days with omega-9 and induced sepsis by cecal ligation and puncture (CLP). We measured systemic corticosterone levels, cytokine production, leukocyte and bacterial counts in the peritoneum, and the expression of PPAR gamma in both liver and adipose tissues during experimental sepsis. We further studied omega-9 effects on leukocyte rolling in mouse cremaster muscle-inflamed postcapillary venules and in the cerebral microcirculation of septic mice. Here, we demonstrate that omega-9 treatment is associated with increased levels of the anti-inflammatory cytokine IL-10 and decreased levels of the proinflammatory cytokines TNF-α and IL-1ß in peritoneal lavage fluid of mice with sepsis. Omega-9 treatment also decreased systemic corticosterone levels. Neutrophil migration from circulation to the peritoneal cavity and leukocyte rolling on the endothelium were decreased by omega-9 treatment. Omega-9 also decreased bacterial load in the peritoneal lavage and restored liver and adipose tissue PPAR gamma expression in septic animals. Our data suggest a beneficial anti-inflammatory role of omega-9 in sepsis, mitigating leukocyte rolling and leukocyte influx, balancing cytokine production, and controlling bacterial growth possibly through a PPAR gamma expression-dependent mechanism. The significant reduction of inflammation detected after omega-9 enteral injection can further contribute to the already known beneficial properties facilitated by unsaturated fatty acid-enriched diets.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/fisiopatologia , Ácido Oleico/farmacologia , Sepse/fisiopatologia , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Modelos Animais de Doenças , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Camundongos , Azeite de Oliva/químicaRESUMO
Neutrophils are the first cells of our immune system to arrive at the site of inflammation. They release cytokines, e.g., chemokines, to attract further immune cells, but also actively start to phagocytose and kill pathogens. In the case of sepsis, this tightly regulated host defense mechanism can become uncontrolled and hyperactive resulting in severe organ damage. Currently, no effective therapy is available to fight sepsis; therefore, novel treatment targets that could prevent excessive inflammatory responses are warranted. Src Family tyrosine Kinases (SFK), a group of tyrosine kinases, have been shown to play a major role in regulating immune cell recruitment and host defense. Leukocytes with SFK depletion display severe spreading and migration defects along with reduced cytokine production. Thus, we investigated the effects of dasatinib, a tyrosine kinase inhibitor, with a strong inhibitory capacity on SFKs during sterile inflammation and polymicrobial sepsis in mice. We found that dasatinib-treated mice displayed diminished leukocyte adhesion and extravasation in tumor necrosis factor-α-stimulated cremaster muscle venules in vivo. In polymicrobial sepsis, sepsis severity, organ damage, and clinical outcome improved in a dose-dependent fashion pointing toward an optimal therapeutic window for dasatinib dosage during polymicrobial sepsis. Dasatinib treatment may, therefore, provide a balanced immune response by preventing an overshooting inflammatory reaction on the one side and bacterial overgrowth on the other side.
Assuntos
Dasatinibe/farmacologia , Infiltração de Neutrófilos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Sepse/imunologia , Animais , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Quinases da Família src/antagonistas & inibidoresRESUMO
The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit(+)/CXCR-4(+) cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases.
Assuntos
Células Endoteliais/metabolismo , Mastócitos/metabolismo , Mecanotransdução Celular , Neovascularização Fisiológica/genética , Neutrófilos/metabolismo , Remodelação Vascular/genética , Animais , Artérias/metabolismo , Artérias/patologia , Plaquetas/citologia , Plaquetas/metabolismo , Proliferação de Células , Células Endoteliais/citologia , Regulação da Expressão Gênica , Membro Posterior/irrigação sanguínea , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Mastócitos/citologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Monócitos/citologia , Monócitos/metabolismo , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Neutrófilos/citologia , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Estresse Mecânico , Linfócitos T/citologia , Linfócitos T/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.
Assuntos
Fator de Crescimento de Hepatócito/imunologia , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Vesículas Secretórias/imunologia , Migração Transendotelial e Transepitelial/imunologia , Músculos Abdominais/irrigação sanguínea , Músculos Abdominais/imunologia , Animais , Membrana Basal/imunologia , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Mucosa Gástrica/química , Mucosa Gástrica/imunologia , Fator de Crescimento de Hepatócito/genética , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Integrina alfa3beta1/genética , Integrina alfa3beta1/imunologia , Integrina alfa6beta1/genética , Integrina alfa6beta1/imunologia , Elastase de Leucócito/genética , Elastase de Leucócito/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Vesículas Secretórias/genética , Migração Transendotelial e Transepitelial/genética , Vênulas/imunologia , Proteínas de Transporte VesicularRESUMO
Mobilization of neutrophils from the bone marrow determines neutrophil blood counts and thus is medically important. Balanced neutrophil mobilization from the bone marrow depends on the retention-promoting chemokine CXCL12 and its receptor CXCR4 and the egression-promoting chemokine CXCL2 and its receptor CXCR2. Both pathways activate the small guanosine triphosphatase Rac, leaving the role of this signaling event in neutrophil retention and egression ambiguous. On the assumption that active Rac determines persistent directional cell migration, we generated a mathematical model to link chemokine-mediated Rac modulation to neutrophil egression time. Our computer simulation indicated that, in the bone marrow, where the retention signal predominated, egression time strictly depended on the time it took Rac to return to its basal activity (namely, adaptation). This prediction was validated in mice lacking the Rac inhibitor ArhGAP15. Neutrophils in these mice showed prolonged Rac adaptation and cell-autonomous retention in the bone marrow. Our model thus demonstrates that mobilization in the presence of two spatially defined opposing chemotactic cues strictly depends on inhibitors shaping the time course of signal adaptation. Furthermore, our findings might help to find new modes of intervention to treat conditions characterized by excessively low or high circulating neutrophils.
Assuntos
Medula Óssea/enzimologia , Neutrófilos/enzimologia , Transdução de Sinais/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Camundongos , Camundongos Knockout , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid ß2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo.